Synthesis of tryptophane and related compounds



Patented July 25 1950 SYNTHESIS OF TRYPTOPHANE AND RELATED COMPOUNDSOwen A. Moe and Donald T. Warner, Minneapolis, Minn., assignors toGeneral Mills, Inc., a corporation of Delaware No Drawing. ApplicationJuly 3, 194 8,

Serial No. 37,065

The present invention relates to the synthesis of tryptophane and torelated amino acids.

Various syntheses for tryptophane have been available. These haveinvolved cumbersome tedious reactions and have entailed considerableexpense, such that the resultant product had to be sold at a very highprice. It has now been discovered thattryp-tophane may be synthesizedfrom readily available materials such that the product may be producedat a reasonable price.

It is therefore an object of the present invention to provide a novelprocess of synthesizing tryptophane and related compounds.

The present application is a continuation-inpart of our copendingapplication, Serial No. 648,020, entitled Aldehydo Compounds andProcesses of Producing the Same, filed February 15, 1946, now abandoned.That application discloses theproduction of various aldehydo compoundsas well as the synthesis of tryptophane from such aldehydo compounds.The aldehydo compounds disclosed have the following structural formula:

R1 lat-R:

in which R is hydrogen, carboxyl, carboxylic acid ester, carbonamide(CONH2, CONHR etc.), or nitrile; R is carboxyl, carboxylic acid ester,carbonamide or nitrile; and R is a substituted amino group in which oneor both of the hydro gens have been replaced. These aldehydo compoundsmay be prepared in a variety of ways, one of which involves the 1,4addition of various malonic esters to alpha,beta-unsaturated aldehydes.For example, the production of gammaacetamido-gamma,gamma-dicarbethoxybutyraldehyde results from the 1,4 addition of ethyl acetamido malonateto acrolein in the presence of an alkaline condensation catalyst.

According to the present invention these aldehydes may be converted totryptophane by reacting them with phenylhydrazine to produce thephenylhydrazone, cyclicizing the phenylhydrazone to form thesubstituted'indole and then hy- 5 Claims. (Cl. 260319) CHa-J'J-COOEL lThe description of the invention is withparticu- 2o lar reference to theemployment of gamma-acetamido-gamma,gamma dicarbethoziy butyraldehydeinasmuch as this is the most convenient and readily available materialto employ. It is to be understood, however, that the invention is notlimited't'o this particular material, as will be apparent more fullyhereinafter. f

The above aldehyde ispr'eferably prepared by the 1,4 addition ofacetamido malonic ester to acrolein vThis reaction is preferably carriedout 40 as follows: The acetamido m'alonic ester is dissolved in'asolvent diluent and a small amount of an alkalinecatalyst is added.Thereaften'the acrolein is added dropwise, and preferably thetemperature is maintained below 50 C. Under these circumstances, a 1,4addition is obtained metallic sodium may be reacted with a large volumeof absolute ethyl alcohol to produce a solu tion of sodium ethoxide inalcohol, the sodium ethoxide being used as the alkaline condensationcatalyst. To this resultant solution there may be added acetamidomalonic ester to produce a mixture which is then preferably cooled. Theacrolein may then be added slowly to this mixture and the temperaturecontrolled to any desired range. The reaction mixture may then beallowed to stand for the required period for the reaction, after whichthe catalyst may be neutralized by means of glacial acetic acid.

In the preparation of tryptophane, it is not essential that the aldehydebe isolated. The reaction mixture containing the desired aldehyde may beconverted directly to the phenylhydrazone. This may be accomplished byacidifying the reaction mixture slightly with glacial acetic acid andadding thereto phenylhydrazine, which results in the formation of thephenylhydrazone. The cyclization of the phenylhydrazone may beaccomplished by adding concentrated sulfuric acid and heating thereaction mixturaior example to reflux temperature, for several hours,after which the reaction mixture is cooled to precipitate a smallquantity of ammonium sulfate which may be removed by filtration. Thefiltrate is then concentrated and cooled to yield a precipitate of theethyl-alpha-acetamido-alpha-carbethoxy-beta-(tl-indole)-propionate. Thissubstituted propionate may then be decarboxylated and hydrolyzed byrefluxing for several hours with an aqueous alkaline solution. Thereaction mix ture is then neutralized to produce alpha-acetamidoalpha-carbethoxy-beta-(3-indole) propionic acid. The reaction mixture isthen cooled to precipitate as much of the propionic acid as possible.

It is thus possible to carry on the entire process from the addition ofacrolein to the ethyl acetamido malonate to the point at which thecyclicized product is obtained without isolation of intermediateproducts. Yields by this process are not as large as are obtainable whenthe reaction is carried to the point of formation of thephenylhydrazone, and isolation effected at this point. It is usuallypreferable, therefore, to carry the reaction without isolation to theformation of the phenylhydrazone, to isolate the phenylhydrazone, toconvert it to the indole propionic ester, to isolate this ester and thento carry on the hydrolysis and decarboxylation of the ester totryptophane in a separate reaction mixture. It will be. apparent,however, that it is possible to isolate the re action product after eachindividual step in the process, if desired.

Excellent results have also been obtained by carrying on the addition ofacrolein to ethyl acetamido malonate in benzene as a reaction solvent,in accordance with the same general process outlined above forthe use ofabsolute ethanol.

Numerous modifications of the cyclization procedure may be employed. Inplace of using suliuric acid in absolute ethanol, it has been found thatimproved results are obtained by using an aqueous ethanol as thereaction solvent or even by employing the heterogeneous phase reactionmixture provided by aqueous sulfuric acid. Surk prisingly enough, evenunder these drastic cyclization conditions hydrolysis of ester groupswas not efiected.

The cyclization reaction may also be accomplished by using borontrifiuoride as the acidic catalyst in an acetic acid reaction solvent.Yields 4 by this procedure are not as satisfactory as with the aqueoussulfuric acid.

The hydrolysis and decarboxylation of the indole propionic acid productmay also be conducted under a variety of conditions. Thus the propionicester may be hydrolyzed with an aqueous alkaline solution to the freepropionic acid, after which the alkaline reaction mixture may beneutralized and the free acid precipitated by cooling. This acid maythen be refluxed with water to decarboxylate the propionic acid andproduce N-acetyl tryptophane which product may be isolated by cooling toprecipitate a white crystalline product. The N-acetyl tryptophane maythen be further hydrolyzed to tryptophane by treatment with an aqueousalkaline solution.

EXAMPLE 1 Preparation of tryptophane using absolute ethanol as a solvent100 parts of absolute ethyl alcohol were treated with-0.05 part ofmetallic sodium. When the reaction of the sodium was complete, 43.7parts of ethyl acetamidomalonate were added. The resultant reactionmixture was a thick slurry, and it Was cooled to 3 C. in an ice bath.Then 12.9 parts of acrolein were introduced dropwise. After the additionof approximately 4 parts of the acrolein, the reaction temperature hadincreased to 13 C. The introduction of the acrolein was interrupted, andthe reaction temperature decreased to 8 C. The remainder of the acroleinwas added at a rate such that the reaction temperature was maintained at8-10 C. After the addition of the acrolein was complete, the reactionmixture was stirred for an additional 30 minute period. The solution wasclear and light brown in color. During the addition of the acrolein, thesolid initially present disappeared. This reaction appeared to proceedin an extremely smooth fashion. The clear, light-colored solution wascooled in an ice bath for an additional hour after which the catalystwas neutralized by the addition of .5 parts of glacial acetic aciddissolved in a small amount of ethanol. The pH of the reaction mixtureat this point was between 4 and 6. The resultingmixture was placed in anice box overnight. After standing overnight the solution was filteredand the solvent was removed by evaporation in vacuo, The residual oilwas very clear and possessed a light yellow-brown color. This wasgamma-acetamido-gamma,gamma-dicarbethoxy butyraldehyde.

Fourteen grams of the crude gammagammadicarbethoxy-gamma-acetamidobutyraldehyde were dissolved in 40 ml. of absolute ethyl alcohol. Threegrams of glacial acetic acid were added. Nine grams of phenylhydrazinewere added to the reaction mixture and the clear solution was warmed to50 C. The mixture was allowed to slowly cool to room temperature andafter standing, the phenylhydrazone ofgamma,gamma-dicarbethoxy-gamma-acetamido butyraldehyde began toprecipitate as light yellow crystals (melting point l38-40 C.). Thecrystals thus precipitated were collected by filtration. The filtratewas diluted with water and yielded an additional quantity of thephenylhydrazone in a somewhat crude form.

Thirty-three parts of the phenylhydrazone of gamma,gamma-dicarbethoxy-gamma-acetamido butyraldehyde were dissolved in 62.4.parts of absolute ethyl alcoholcontainin'g one part ofjwater 5. andparts of concentrated sulfuric acid. The reaction mixture was heateduntil the reflux temperature was attained and the reflux temperature wasmaintained for a period of 2 hours. The reaction mixture was cooled toroom temperature and placed in a refrigerator overnight. A small amountof ammonium sulfate precipitated from the clear amber liquid. Theammonium sulfate was removed by filtration and th filtrate, upon furtherconcentration and cooling, yielded a copious precipitate. Thisprecipitate was collected by filtration, washed with absolute ethylalcohol, and a white crystalline solid was obtained (melting point154-156 C.). Further concentration of the filtrate yielded additionalquantities of this solid (melting point l50- 155 C.). Finally, thealcoholic filtrate was diluted with an equivalent quantity of water andthe product thus precipitated was collected by filtration. A substantialquantity of this crude material was obtained and it melted at 110-120 C.The white crystalline solid mentioned above wasethyl-alpha-acetamido-alpha carbethoxy beta-(3-indole) -propionate.

3.4 parts of ethyl-alpha-acetamido-alpha-carbethoxy-beta-(3-indole)-propicnate were refluxed for 2 and hours with an aqueous solution ofsodium hydroxide containing 1.92 parts of sodium hydroxide dissolved in19.2 parts of water. A light brown solution resulted. This solution wasdecolorized with about 0.1 part of carbon black and a clear, lightyellow solution resulted. This alkaline solution was cooled in an icebath and neutralized slowly with 5.9 parts of concentrated hydrochloricacid. The temperature of the reaction mixture was kept below C. duringthe addition of the concentrated acid. A copious precipitate ofalpha-acetamido-alpha-carboxy-beta- (3-indole) -propionic acid wasformed. After cooling in the ice bath for 1 hours, the light pinkprecipitate was collected by filtration. It was dried in a vacuumdesiccator over calcium chloride for 16 hours. The product thus obtainedmelted at 141-143 C. (decomposition).

2.8 parts of alpha-acetamido-alpha-carboxybeta-(S-indole)-propionic acid(melting point: 141-143 C. with decomposition) were mixed with 12 partsof water. The resulting aqueous mixture was refluxed for a period of 2hours. Some separation of the N-acetyl tryptophane was observed as therefluxing proceeded. The decarboxylation appeared to be substantiallycomplete after two hours, and the reaction mixture was cooled. Uponcooling, a white crystalline product was deposited. The crystallineproduct was collected by filtration and dried. This N-acetyl tryptophanemelted at 202-204 C.

1.4 grams of N-acetyl tryptophane (melting point 202-204 C.) weredissolved in aqueous sodium hydroxide solution containing 1.5 g. ofsodium hydroxide in 15 g. of water. The resulting reaction mixture washeated until the reflux temperature was attained. The reflux temperaturewas maintained for a period of 7-14 hours in an oil bath. The reactionmixture was then cooled and neutralized by the gradual addition of 2.4g. of glacial acetic acid. As the addition of the glacial acetic acidprogressed, a copious white precipitate was noted. The reaction mixturewas chilled in an ice bath and the precipitated product was collected byfiltration. The crude dltryptophane thus obtained melted at 255-263 C.(decomposition). Further purification of the material increased themelting point to 270-280 C. with decomposition.

ma-dicarbethoxy-butyraldehyde using benzene as the reaction solvent Theethyl acetamidomalonate (217 g.) was suspended in a benzene solution(330 cc.) containing a catalytic quantity of sodium methoxide (0.5 g.).The resulting reaction mixture was cooled in a water bath at 19 C.Acrolein (68.5 cc.) dissolved in benzene (70 cc.) was added dropwise ata moderate rate. The temperature increased to 35 C. and after theaddition of the alpha,beta unsaturated aldehyde was complete, thereaction mixture was stirred for an additional two-hour period. Afterneutralization by the addition of the requisite quantity of glacialacetic acid (1 00.). the reaction mixture was filtered and the clearlight yellow filtrate was treated with glacial acetic acid (23 cc.) andphenylhydrazine g.). The reaction mixture was warmed to 50 C. and theresulting solution was permitted to stand at room temperature for aperiod of three days. The phenylhydrazone was collected by filtrationand washed with 150 cc. of benzene. After suspension in 250 cc. ofbenzene, the phenylhydrazone was collected and dried in vacuo. The yieldof the phenylhydrazone ofgamma-acetamido-gamma,gamma-dicarbethoxybutyraldehyde was 315.1 g. (87%)melting at -41 C., and it was nearly white in color.

EXAMPLE 3 Cyclization reaction using concentrated sulfuric acid in 50volume percent aqueous ethanol Fifty grams (50 g.) of thephenylhydrazone of gamma-acetamido-gamma,gammadicarbethoxy butyraldehydewere mixed with 50 volume percent aqueous ethanol (62.5 cc. absoluteethanol and 62.5 cc. of water) containing 9 cc. of concentrated H2804.The resulting reaction mixture was refluxed for a seven-hour period. Aclear solution was maintained throughout the entire reflux period. Theresulting reaction mixture was cooled in the refrigerator overnight, andthe crystalline product was collected by filtration, washed with waterand dried in vacuo. The yield of ethyl alpha acetamidoalpha-carbethoxybeta-(3-indole)-propionate melting at 153-4 C. was 28.8g. which represents a 60% yield.

EXAMPLE 4 Cyclization reaction using concentrated sulfuric acid in 40volume percent aqueous ethanol When the cyclization reaction was carriedout with 40 volume percent aqueous ethanol using the same quantities ofmaterial as noted in Example 3, a clear solution was obtained as soon asthe reflux temperature was reached. However, after about one hour thecyclicized product precipitated from the reaction mixture as an oil. Theprecipitated oil solidified on cooling, and the yield of the cyclicizedproduct was comparable to that obtained in Example 3.

EXAMPLE 5 Cyclie'ation reaction involving aqueous sulfuric acid Fiftygrams of the phenylhydrazone of gammaacetamido-gamma,gamma dicarbethoxybutyraldehyde were mixed with 300 cc. of water containing 14 cc. ofconcentrated sulfuric acid. The resulting reaction mixture was heated tothe reflux temperature with very vigorous stirring. The phenylhydrazoneappeared to-liquofy at I: the reflux temperature, andafter approximatelyone hour the liquid suspension was transformed to a solid suspension.The reflux temperature was maintained for 'a period of four and onehalfhours. After cooling, the solid reaction product was collected and thenmixed with water in a mixer. After filtration and washing with water,the desired product was dried in vacuo. The yield of oyclicized productobtained was 42.5 g. representing a 96% yield, and it melted at 145-9 C.Recrystallization from aqueous ethanol (50-50) gave 35 g. (73% yield) ofethyl-alphaacetamido-alpha-carbethoxy-beta-(3 indole) propionate meltingat 155-7 C.

EXAMPLE 6 C'yclzeation reaction employing aqueous sulfuric acid Twohundred seventy grams (270 g.) of the phenylhydrazone ofgamma-acetamido-gam-ma,- gamma-dicarbethoxy butyraldehyde were mixedwith 1500 cc. of water containing 129 g. of concentrated sulfuric acid.The resulting reaction mixture was agitated vigorously as it was heatedto the reflux temperature. After the reflux temperature had beenattained, the solid phenylhydrazone was transformed to a liquid productwhich was finely dispersed in the aqueous phase. Within an hour theliquid product had been changed to a finely divided solid. The refluxtemperature was maintained for a period of three hours. The reactionmixture was then cooled to room temperature and the solid cyclicizedproduct was collected by filtration and washed with two liters of coldwater. Ihe reaction product was then mixed with water in a mixer,collected by filtration, washed with water and dried in vacuo. The drycyclicized product weighed 229 g. and melted at 151-3 C. It was obtainedas a light tan amorphous powder. After recrystallization from aqueousethanol (50-50), 177 g. of ethyl-alpha acetamido alphacarbethoxybeta-(3-indole) -propionate melting at 156-7 C. was obtained.

EXAMPLE 7 C'yclizatzon reaction employing boron triflaorzde as theacidic catalyst Thirty-six and three-tenths grams (36.3 g.) of thephenylhydrazone f gamma-acetamido-gamma,gamma-dicarbethoxy butyraldehydewere mixed with 100 cc. of glacial acetic acid. Fourteen and two-tenths(14.2 g.) of boron trifluoride etherate were added with swirling and theresulting reaction mixture was heated cautiously in an oil bath. Thereflux temperature was maintained for a period of thirty minutes. A

copious precipitate of the boron trifiuoride-ammonia complex was noted,and after cooling it was removed by filtration. Water was added to thefiltrate to yield a slight turbidity and the reaction mixture was cooledin the refrigerator overnight. The precipitated product (amorphous inappearance) was collected by filtration and dried in vacuo. The driedproduct weighed 13.8 g. and melted at l37-l2 C. Recrystallization from50 volume percent aqueous ethanol yielded 8.8 g. yield)ethyl-alpha-carbethoxy-alphaacetamido-beta-(3-indole) -propionate.-at153 C.

While numerous modifications of the invention have been described, othermodifications are also possible without departing from the spirit of theinvention.

We claim. as our invention: 7 1. Process. of producing compounds havingthe formula R CHa-JI-R r' where R is carboxyalkyl; R is selected fromthe group consisting of carboxya'lkyl and nitrile; and R is an acylaminogroup, which comprises reacting phenylhydrazine with a compound havingthe formula Rx lathe I E0 to form the phenylhydrazone, and cyclicizingthe phenylhydrazone in the presence of aqueous sulfuric acid as thecatalyst at reflux temperatune to form the first mentioned compound.

3. Process of producing compounds having the to form thephenylhydrazone, and cyclicizing the .phenylhydrazone in the presence ofa mixture of. ethanoland'sulfuric acid as catalyst at refluxtemperatureto formthe first mentioned compound. a

A e mu.

4. Process of producing compounds having the formula R CHr--R where R iscarboxyalkyl; R is selected from the group consisting of carboxyalkyland. nitrile; and R is an acylamino group, which comprises reactingphenylhydrazine with a compound having the formula.

to form the phenylhydrazone, and cyclicizing the phenylhydrazone in thepresence of dilute aqueous sulfuric acid as the catalyst at refluxtempera-- ture to form the first mentioned compound.

5. Process of producing ethyl-alpha-acetamidoalpha carbethoxy beta (3indole) propionate, which comprises reacting phenylhy- REFERENCES CITEDThe following references are of record in the file of this patent:

UNITED STATES PATENTS Name Date Gassner May 1, 1928 Meig et a1. Sept.11, 1928 Snyder et a1. Aug. 24, 1948 Snyder et a1. Aug. 24, 1948 OTHERREFERENCES Ellinger: Berichte Deut. Chem. Gesell, 38 (1905), 2884-2888.

Snyder et a1.: Jour. Am. Ch. 800., 66 (March 1944), 350-351.

Albertson et a1.: Jour. Am. Ch. Soc., 66 (March 1944), 500.

Number Certificate of Correction Patent No. 2,516,332 July 25, 1950 OWENA. MOE ET AL.

It is hereby certified that error appears in the printed specificationof the above numbered patent requiring correction as follows:

Column 7, line 55, after two-tenths insert the Word grams; line 70,after (25% yield) insert of; line 71, after propionate insert melting;same line, for 153 C. read 1535 0.; and that the said LettersPatentshould be read as corrected above, so that the same may conform tothe record of the case in the Patent Oflice.

Signed and sealed this 17th day of October, A. D. 1950.

THOMAS F. MURPHY,

Assistant Commissioner of Patents.

1. PROCESS OF PRODUCING COMPOUNDS HAVING THE FORMULA